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Montara Well Release Monitoring Study S4A Phase IV

Assessment of Effects on Timor Sea Fish Final Report

MONITORING STUDY S4A (PHASE IV)

FINAL REPORT

Summary of Results

• Phase IV of the monitoring study conducted two years following the Montara well release collected biological parameters from 486 goldband snapper (Pristipomoides multidens) and 395 red emperor (Lutjanus sebae) collected over 13 sampling locations.

• For both species of fish, all individuals appeared in good physical condition.

• Goldband snapper were of similar body length at all sites. Red emperor collected close to the well head were smaller in size relative to those from other sites, most likely due to the fishing pressure which this local fish population has experienced in the past two years.

• For both species of fish, individuals collected close to the wellhead had larger livers relative to body size, compared to fish collected at the reference location.

• Biomarkers of exposure to petroleum hydrocarbons showed that in the fish collected close to the well head, no recent exposure had occurred in the recent weeks prior to the sampling.

• As in the past samplings, fish collected within the Heywood Shoal area exhibited elevated biomarker responses indicating recent exposure to petroleum hydrocarbons. Fish returning positive biomarker responses were collected at a site close to the Cornea natural seep.

• Maturation of gonadal tissues was progressing in similar stages at impacted and reference sites.

• Overall, fish collected at the four sites closest to the rig, all located within 27 NM from the well head, showed no recent exposure to petroleum hydrocarbons. Biochemical markers measured in fish collected close to the well head showed that biomarkers of exposure to petroleum hydrocarbons measured in individuals collected in the area most impacted by the Montara well release have returned to reference levels. The liver size relative to body size in the fish collected close to the well head was larger compared to fish originating from a reference site however, this could be related to local nutrient enrichment, or to past exposure to petroleum hydrocarbons.

• Future monitoring will indicate if liver somatic index in fish collected close to the well head shows a trend towards return to reference conditions, or if the observation results in a permanent condition related to natural environmental conditions present at these sites.

• Phase IV of the monitoring program has provided valuable baseline data for future monitoring of produced formation water discharge when operations commence at the offshore facility.


FINAL REPORT

Preface This report was prepared by Marthe Monique Gagnon and Christopher Rawson from the Department of Environment and Agriculture, Curtin University. The sampling event recorded herein occurred aboard the FV Megan M between the 16th of October and the 7th November 2011.

Acknowledgements Special thanks to the skipper (Grant Barker) and crew (Lindsay Smith and Mitch Seelander) of the FV Megan M for their assistance in the collection of fish samples. Thanks to Tomoe Ota for the careful processing and examinations of gonadal tissues.

Document History

Rev Date Description Prepared by Comments

provided by

A 3 Aug 2012 Draft report submitted to

PTTEP

MM Gagnon

CA Rawson

B 22 Aug 2012 Comments provided G. Nicholson*

C 14 Sept 2012 Final Report submitted to

PTTEP

MM Gagnon

CA Rawson

* See Appendix I for Comments and Author’s responses

For further information contact: Associate Professor Marthe Monique Gagnon Department of Environment and Agriculture Curtin University Perth, Western Australia, 6845 Tel.: (08) 9266 3723 Email: [email protected] Recommended Citation: Gagnon M.M., Rawson C., 2012. Montara Well Release, Monitoring Study S4A Phase IV – Assessments of Effects on Timor Sea Fish. Curtin University, Perth, Western Australia. 66pp.

Disclaimer The views contained in this report are those of the authors and do not necessarily reflect the views of PTTEP Australasia, SEWPaC, or any other party.

ISBN: 978-0-9872223-2-9


FINAL REPORT

Curtin University was contracted by PTTEP Australasia to conduct a fish monitoring study

on demersal and pelagic fishes in the Timor Sea, in waters affected by the Montara well

release. In November 2011 Associate Professor Marthe Monique Gagnon and Dr.

Christopher Rawson conducted 3 weeks of field sampling aboard the FV Megan M

commercial fishing vessel, collecting biopsies of serum, liver, gonadal tissue and bile for

laboratory biomarker analysis. This study was the fourth such study following the well

release and utilised the same sites as earlier phases of the program with 5 additional sites

to the north-west and south east of the Montara well head.

The sampling event targeted the commercially important demersal species goldband

snapper (Pristipomoides multidens) and red emperor (Lutjanus sebae). A total of 881

demersal fish were captured during the study with biopsies taken on 519 fish.

The selected physiological indicators included the condition factor, liver somatic index, and

gonado-somatic index. Biochemical markers selected for their relevance to exposure to

petroleum hydrocarbons were liver detoxification enzymes, biliary polycyclic aromatic

hydrocarbon metabolites, liver integrity, and oxidative DNA damage. In addition,

histological examination of the gonads was performed.

Executive Summary


FINAL REPORT

Previous studies conducted in the areas affected by the Montara incident showed that one

year following the hydrocarbon well release, physiological parameters as well as

biochemical markers of fish health showed a return towards reference levels for fish

captured close to the rig location, with the exception of liver size which varied between

impacted and reference areas, and a few fish collected at one impacted site (Heywood

Shoal) which showed continuing evidences of exposure to petroleum hydrocarbons.

Phase IV of the study was conducted 24 months following the end of the hydrocarbon

release. Goldband snapper collected at the sites closest to the rig were of similar size to

those originating from a reference site, while red emperor collected close to the rig were of

smaller size relative to their counterpart collected from a reference site. While goldband

snapper has a relatively rapid growth rate and did not show population size differences,

red emperor is a slow growing fish. This smaller size observed in red emperor collected

close to the rig is likely due to the recent fishing pressure experienced by the population at

this location, combined with the slow growth rate of this fish species. While the physical

condition of the fish of both species was good at all sites, the liver size relative to body size

of both species of fish remained elevated at the sites closest to the rig. However, other

biomarkers of exposure such as liver detoxification enzymes, PAH biliary metabolites and

oxidative DNA damage were comparable in fish collected close to the rig and in fish

originating from the reference site, confirming that uptake of petroleum hydrocarbons has

not occurred in the recent weeks. For male and female individuals of both species, the

gonad size relative to body size of adult fish collected close to the rig were similar to those

collected at the reference site as were the stage of egg maturation within the female

gonads.

Of particular interest were the fish collected in the vicinity of Heywood Shoal. In previous

studies a few individuals returned positive biomarkers of exposure to petroleum

hydrocarbons, suggesting the possibility of exposure to natural seepages in the area. In

Phase IV, four locations in the vicinity of Heywood Shoal were discretely sampled to

identify if these biomarkers of exposure were still high two years following the incident.

Male red emperor exhibited a significant liver detoxification activity at one of the sites,

suggestive of exposure to hydrocarbons. Female red emperor from the same site had liver

detoxification activity similar to those fish from the reference area (female fish frequently

have hormonally-inhibited liver detoxification systems). Red emperor collected at the three

other sites close to Heywood Shoal, as well as all collected goldband snapper, had liver

detoxification activity similar to those fish originating from the reference site. Biomarker


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induction in male red emperor collected in the area of Heywood Shoal is consistent with

past observations made at this site. It has been noted that the Cornea seep is in proximity

to one of the Heywood Shoal sampling sites, and might contribute to the ongoing high

biomarker responses observed at Heywood Shoal. .


FINAL REPORT

Table of Contents

Executive Summary ........................................................................................... 4

List of Figures .................................................................................................... 9

List of Tables .................................................................................................. 12

Background .................................................................................................... 13

Project Aims (Phase IV) .............................................................................................. 15

Sampling Timeline .......................................................................................... 16

Sampling Sites ............................................................................................... 19

Fish Sampling................................................................................................. 23

Morphology ................................................................................................................. 23

Biopsies Collected ...................................................................................................... 24

Gonad Samples for Histology ...................................................................................... 25

Additional Sampling .................................................................................................... 25

Additional Samples ..................................................................................................... 25

Laboratory Methods ....................................................................................... 26

Physiological Parameters ............................................................................................ 26

Liver Detoxification Enzymes (EROD activity) .............................................................. 27

Biliary Metabolites ....................................................................................................... 27

Sorbitol Dehydrogenase (SDH) activity ........................................................................ 28

Gonad Histology ......................................................................................................... 28

Oxidative DNA Damage (8-oxo-dG Content) ............................................................... 29

Statistical Analyses ..................................................................................................... 29

Results & Interpretation ................................................................................. 31

Fish Sampled .............................................................................................................. 31

Biopsy Collection ........................................................................................................ 33

General Fish Health .................................................................................................... 34

Physiological Parameters ................................................................................ 34

Condition Factor.......................................................................................................... 35

Liver Somatic Index..................................................................................................... 37

Gonado-somatic Index ................................................................................................ 40

Gonad Histology ......................................................................................................... 43

Biochemical Parameters ................................................................................. 47

Liver Detoxification Enzymes ...................................................................................... 47


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Biliary Metabolites ....................................................................................................... 51

Sorbitol Dehydrogenase (SDH) Activity ....................................................................... 54

DNA damage .............................................................................................................. 56

Conclusions.................................................................................................... 58

Recommendations ......................................................................................... 61

References..................................................................................................... 63

Appendix I – Comments and Authors’ Responses ............................................ 66


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List of Figures

Figure 1. Map showing the location of the Montara well head and the sampling sites.

Concentric rings represent 20, 80 and 150 NM from the well head. ................. 21

Figure 2. Method of demersal fish capture. Baited steel fish trap prior to deployment. In

picture: Matthew Badart (left) and Beau Pieterman (right). .............................. 24

Figure 3. Parasites commonly found in the abdominal cavity of goldband snapper and

red emperor..................................................................................................... 34

Figure 4. Condition factors of goldband snapper and red emperors collected at the

various study sites during Phase IV of the monitoring study S4A. For both

species, fish from all sites had statistically similar CF (p > 0.05 in all cases). .. 36

Figure 5. Liver somatic index in goldband snapper and red emperors captured at study

sites in the Timor Sea in November 2011. Bars with different letters indicate

statistical differences. Sites 1, 2, 14, 15 were all located within 27 NM of the

hydrocarbon release and were considered impacted, while reference Site 5 was

located at 110 NM from the West Atlas rig location. LSI at the sites close to

thedrill rig was different from LSI in reference fish (p < 0.000). ........................ 39

Figure 6.Gonado-somatic index of male and female goldband snapper captured at

study sites in the Timor Sea in Phase IV of the monitoring. Bars with different

letters indicate statistical differences. Fish captured at sites within 27 NM of the

well head (sites 1, 2, 14, 15) have similar GSI relative to fish from the reference

Site 5. GSI in male fish originating from Site 12 is different from GSI in male fish

from the reference Site 5 (p =- 0.006). GSI measured in female fish collected at

Site 10 is different from GSI measured in female fish from the reference Site 5 (p

= 0.001). .......................................................................................................... 41

Figure 7. Gonado-somatic index of male and female red emperors captured at study

sites in the Timor Sea in Phase IV of the monitoring. Male and female fish

captured at sites within 27 NM of the well head (sites 1, 2, 14, 15) had similar

GSI relative to fish from the reference Site 5 (p > 0.05 in all cases)................. 42

Figure 8. Histological (HE stain) examination of intersex gonad of morphologically

male goldband snapper collected from Site 13. Inset shows characteristic lobed

structure of male testis (note the mature sperm in the efferent duct). ED =


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Efferent Duct, S = Sperm (with developmental stage indicated), O = oocyte (with

developmental stage indicated). ...................................................................... 44

Figure 9. Percent composition of ovaries of goldband snapper and red emperor from

Timor Sea study sites by oocyte stage. Oocyte stages defined and described

given in Table 2. .............................................................................................. 46

Figure 10. EROD activity (pmol/mg protein/min) in male and female goldband snapper

captured as study sites in the Timor Sea in November 2011. EROD activity in

fish collected within 27 NM of the rig (sites 1, 2, 14, 15) is not statistically

different (p = 0.419 and 0.700 for male and female, respectively) form EROD

activity in fish from the reference Site 5. Other investigated sites were not

statistically different from the reference Site 5. ................................................ 49

Figure 11. EROD activity (pmol/mg protein/min) in male and female red emperor

captured as study sites in the Timor Sea in November 2011. Bars with different

letters indicate statistical differences. EROD activity in fish collected within 27

NM of the rig (sites 1, 2, 14, 15) is not statistically different (p = 0.169 and 0.605

for male and female, respectively) form EROD activity in fish from the reference

Site 5. Site 8 is also significantly different form the reference Site 5 (p < 0.001

and p = 0.003 for male and female respectively). ............................................ 50

Figure 12. Levels of naphthalene- (mg/mg protein), pyrene- (ug/mg protein) and BaP

(ug/mg protein) -type biliary metabolites in goldband snapper captured at study

sites in the Timor Sea in November 2011. Bars with different letters indicate

statistical differences. Goldband snapper collected from study sites located

within 27 NM from the well head had similar biliary metabolite levels (p =0.056,

0.062 and 0.454 for naphthalene, pyrene and BaP-type metabolites respectively)

relative to fish originating from reference Site 5. .............................................. 52

Figure 13. Levels of naphthalene- (mg/mg protein), pyrene- (ug/mg protein) and BaP

(µg/mg protein) -type biliary metabolites in red emperor captured at study sites in

the Timor Sea in November 2011. Red emperor collected from study sites within

27 NM from the well head had similar biliary metabolite levels (p =0.166, 0.522

and 0.872 for naphthalene, pyrene and BaP-type metabolites respectively)

relative to the fish collected at the reference Site 5.......................................... 53

Figure 14. SDH activity (mIU) in goldband snapper and red emperor collected from

study sites in the Timor Sea in November 2011. Both species of fish collected in

the vicinity of the rig (sites 1, 2, 14 15) had comparable SDH activity levels to the


FINAL REPORT

fish collected at the reference Site 5 ( p = 0.330 and 0.101 for goldband snapper

and red emperor respectively). ........................................................................ 55

Figure 15. Oxidative DNA damage (ng/ml/mg protein) in goldband snapper and red

emperor at study sites in the Timor Sea in November 2011. No statistical

differences were identified between goldband snapper collected at the study

sites (p = 0.662). Between site differences were detected between red emperor

collected between sites (p = 0.036) but no differences were detected between

the reference site (Site5) and any of the other study sites. .............................. 57


FINAL REPORT

List of Tables

Table 1. Details of S4A Phase IV sampling sites ...................................................... 22

Table 2. Scale used for determining oocyte stage in goldband snapper and red

emperor. .......................................................................................................... 29

Table 3. Numbers of demersal fishes sampled during Phase IV of Study S4A. ........ 32

Table 4. Numbers of fish from which biopsies were collected during the current phase

of study S4A. ................................................................................................... 33


FINAL REPORT

The Montara incident which commenced on Friday 21st August, 2009, resulted over 74

days in the release of gas, condensate and crude oil into the Timor Sea and released an

estimated 23,000 barrels of oil and gas condensate into the Timor Sea. The release

occurred in an area utilised for commercial fishing activities (Northern Demersal Scale

Fishery). In addition, the area is an ecologically important habitat to a variety of marine life

including fish, reptiles, cetaceans and aquatic birds. Short- and long-term impacts are

possible following exposure to petroleum hydrocarbons, and consequently it is imperative

that any impact of the well release on commercial fish populations be assessed.

The most relevant and sensitive assessment of petroleum hydrocarbon exposure and

impact on fishes is through the use of biochemical markers (biomarkers) of fish health –

biomarkers are sublethal biological endpoints that can be used to quantify exposure or

effect. No single biomarker can give an overall assessment of fish health and

consequently, a suite of biomarkers is measured on each individual fish. These include

measuring the metabolites of polycyclic aromatic hydrocarbons (PAHs) in the bile of the

fish (PAHs are generally not accumulated in fish muscle; Varanasi et al., 1989), estimating

the amount of DNA damage that has occurred, measuring the activity of liver detoxification

enzymes, and measuring liver integrity. In addition, histological examination of the gonads

can give important information on alteration to the reproductive state of the animal as a

result of exposure to the contaminants.

Background


FINAL REPORT

Immediately following the control of the 2009 well release, Curtin University was

contracted by PTTEP Australasia on agreement with the Australian Government

Department of Environment Heritage, Water and the Arts (DEWHA) (now SEWPaC) to

investigate (using biomarkers of fish health) any exposure and/or effects of the well

release on fish health in the Timor Sea. As part of the scientific monitoring program agreed

to by DEWHA two assessments would initially be made to investigate these exposures

and effects and note any changes over the intervening time (Phase I and Phase II).

Samples comprising these assessments were collected in November 2009 and March

2010. Following these initial sampling events a further study (Phase III) was conducted 12

months following the end of the hydrocarbon release. Shortly after the end of the incident,

these studies investigating fish health showed evidences of exposure to petroleum

hydrocarbons at sites close to the West Atlas drilling rig, increased liver size and increased

oxidative DNA damage. One year after the end of the well release however, a reduced

biomarker response suggested an ongoing trend towards a return of biomarkers to

reference levels (Gagnon and Rawson, 2011).

The current study was undertaken as a follow-up to Phase I-III investigations. It collected

samples at 13 sampling sites located between 1 NM and 110 NM from the West Atlas drill

rig. It also performed intensive sampling at one specific location, Heywood Shoal, as

previous investigations showed that a few individual fish collected at this site returned

positive results suggesting continuing exposure to petroleum hydrocarbons.


FINAL REPORT

Project Aims (Phase IV)

• To further characterise any exposure, including the geographical extent of the

exposure, of commercially important Timor Sea demersal fishes to petroleum

hydrocarbons following the Montara well release.

• To evaluate if fish health, including reproductive health, is currently affected by

exposure to petroleum hydrocarbons.

• By re-sampling the same sites as investigated in Phases I - III of the monitoring

program, to investigate any long-term trends in physiological indices and biomarker

levels in commercially important fish species from impacted and reference areas.

• To expand the number of sites studied to gain a more complete understanding of

the biochemical and health status of fish in the Timor Sea, especially at Heywood

Shoal.

• To establish baseline data for future monitoring following the commencement of

PFW discharge at the Montara wellhead.


FINAL REPORT

16th October 2011 Christopher Rawson arrives Darwin (PM). 17th October 2011 Christopher Rawson arranges delivery of field gear from PTTEP

depot to Megan M. 1300hrs: Monique Gagnon arrives Darwin. 18th October 2011 1900 hrs: Megan M departs from Fishermans Wharf, Darwin.

Aboard: Scientific team: Monique Gagnon and Christopher Rawson (Curtin University); FV Megan M skipper Grant Barker; FV Megan M crew Lindsay Smith, Mitchell Seelander. Steam due west.

19th October 2011 Continue steaming toward sampling area. Organisation of field gear

in preparation for scientific sampling. 20th October 2011 1200hrs: Arrive Site 8. Traps set. 1600hrs: traps lifted and scientific

sampling commences. Anchor overnight at Site 8 with traps set for red emperor.

21st October 2011 0600hrs: Traps lifted (full complement of fish captured). 1200hrs

steam toward Site 9. 1400hrs: Traps set at Site 9. 1700hrs: traps lifted and reset for overnight soak.

22nd October 2011 0630hrs: Overnight traps lifted and reset. 1200hrs: traps lifted. (full

complement of fish captured). Steam to Site 15. 2300hrs: arrive Site 15 and set anchor.

23rd October 2011 0600hrs: traps set (Site 15). 1100hrs: traps lifted and reset. 1400hrs:

traps lifted. 1700 steam SE to Site 14. 1930hrs: arrive Site 14 and set anchor.

Methodology

Sampling Timeline


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24th October 2011 0600hrs: Traps set (Site 14). 1100hrs: Traps lifted and reset. 1400hrs: Traps lifted. Steam toward Site 2. 1930hrs: Arrive Site 2 and set anchor.

25th October 2011 0600hrs: Traps set (Site 2) within 2 NM of Montara well head.

1130hrs: traps lifted and reset. 1930hrs: sampling completed. Steam to Site 1.

26th October 2011 0630hrs: Traps set (Site 1). 1130hrs: traps lifted and reset. 1615hrs:

traps lifted (full complement of fish collected). 1800hrs: steam toward Site 13. 2300hrs: Arrive at Site 13 and set anchor.

27th October 2011 0550hrs: traps set (Site 13). 1100hrs: traps lifted. Only goldband

snapper collected at this site (designated 13A) so selected site for red emperor 12NM NE (designated Site 13B). 1230hrs: set traps (Site 13B). 1530 hrs: traps lifted. 1800 hrs: Steam toward Site 12.

28th October 2011 0600hrs: traps set (Site 12). 1100hrs: traps lifted and reset. 1600hrs:

traps lifted and steam toward Site 4. 29th October 2011 0530hrs: traps set (Site 4 – Heywood Shoal). 1030hrs: traps lifted

and reset. 1500hrs: traps lifted and reset. 1800: traps lifted and reset. 1900hrs: steam toward Site 11.

30th October 2011 0630hrs: traps set (Site 10). 1130hrs: traps lifted and reset. 1700hrs:

traps lifted (full complement of fish captured) and steam toward Site 11.

31st October 2011 0600hrs: traps set (Site 11). 1200hrs: traps lifted and reset. 1730hrs:

traps lifted and steam toward Site 16. 1930: Arrive Site 16 and set anchor.

1st November 2011 0515hrs: traps set (Site 16). 1030hrs: traps lifted and reset. 1630hrs:

traps lifted and steam toward Site 5 (Browse Is). 2nd November 2011 0130hrs: arrive Browse Is. and set anchor. 0500hrs: traps set (Site

5). 1100hrs: traps lifted and reset. 1530hrs: traps lifted. Completion of scientific sampling. Steam toward Darwin.

3rd November 2011 Steaming toward Darwin. 4th November 2011 Steaming toward Darwin. 5th November 2011 Steaming toward Darwin. 1400hrs: arrive Darwin Harbour. 1900hrs:

Enter Duckpond through tide lock and disembark FV Megan M. Equipment readied for transport to Perth via PTTEPAA (TNT).


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6th November 2010 0900hrs: Field gear picked up by PTTEPAA (Steve Welch). Cryogenic dry shippers refilled. Frozen samples sent via cold transport.

7th November 2011 Monique Gagnon and Christopher Rawson depart Darwin and arrive

Perth.


FINAL REPORT

The sites from which fish were collected were based on those sampled during Phases I –

III with the addition of extra sites at the request of PTTEP and the deletion of other sites

where only pelagic fishes were previously sampled. When specific sites previously fished

were known to produce either lower catches or smaller fish, traps were generally set at

locations slightly removed from those previously sampled as part of the Montara

monitoring program. The locations of the original sampling sites were selected based on

the information on the extent, location and direction of hydrocarbon sheen and/or surface

(wax) residue (based on AMSA satellite imagery and ship and aircraft observations)

provided by DEWHA and PTTEP Australasia at the time of the S4A Phase I. Based on

information available reference sites were selected not less than 80 NM south west of the

West Atlas drilling platform. Information provided subsequent to the Phase I sampling

suggested that the well release had affected areas outside of this radius. Further, sites

were selected based on water depth (shallow reef <30m, or deep sea > 70m) and sea floor

structure (hard or soft bottom) which allowed for the reasonable expectation of consistency

of catch diversity and numbers (Figure 1, Table 1).

• Site 1 located 20 NM to the SSW of the West Atlas drilling rig with Site 2 located

within 2 NM as in previous phases of the study.

• Site 3 was selected for the sampling of pelagic species during Phase I of the study.

This site was deleted in subsequent sampling events since insufficient numbers

were captured after an extensive sampling effort.

Sampling Sites


FINAL REPORT

• Site 4 was located at Heywood Shoal to the SW of the Montara well head and has

been sampled in all previous sampling events. As a result of previous results

sampling was intensified at this location, with 3 extra sites (Sites 10, 11 and 16)

added to better characterise potential hydrocarbon exposure at this location.

• Sites 5, 6 and 7 were located within the reference area. Sites 6 (Echuca Shoal) and

7 (Scott Reef) were not required in Phase IV as only pelagic fishes were captured

at these sites. In Phase IV, demersal fishes were captured at Site 5 (Browse

Island). This site represents the reference site for demersal fish.

• Sites 8 and 9 were designated as reference sites during a sampling trip conducted

by WA Fisheries in January 2010 and were included in Phases II, III and IV of

study S4A. Examination of satellite imagery however later revealed that these sites

were also impacted by the hydrocarbon slick.

• Sites 12, 13A and 13B to the SE of the Montara well head were added to the

sampling program for Phase IV at the request of PTTEP. These new sites, along

with the existing reference site, will provide background data for future monitoring

once the new production facility is operational.

• Sites 14 and 15 were located to the NW of the Montara well head and were added

in Phase IV of the study. These sites were located in 70 – 120m water and were

sampled for demersal species only.

Figure 1. Map showing the location of the Montara well head and the sampling sites. Concentric rings represent 20, 80 and 150 NM from the well head.


FINAL REPORT

Table 1. Details of S4A Phase IV sampling sites

Site Location Distance from West Atlas

(NM) Lat (o) Long (o)

1 12.9450 S 124.900 E 19

2 12.6545 S 124.5575 E 1

4 Heywood Shoal 13.4090 S 124.1437 E 50

5 Browse Island 14.2422 S 123.5692 E 110

8 WA Fish. Ref 11.9016 S 125.6345 E 80

9 WA Fish Ref 12.1505 S 125.3120 E 55

10 13.4207 S 123.9722 E 56

11 13.5303 S 124.6375 E 52

12 13.3912 S 125.0572 E 53

13A 13.2712 S 124.8492 E 40

13B 13.0985 S 125.1492 E 44

14 12.5017 S 124.3632 E 15

15 12.3003 S 124.2841 E 27

16 13.6228 S 124.5522 E 57


FINAL REPORT

The methods of fish capture were similar to those in S4A Phases I, II and III.

Demersal fishes were collected from 70-100m depth using baited stainless steel fish

traps (Figure 2). The traps were dropped at the sampling locations and left for

between 1 and 12 hours. Two demersal species were targeted: goldband snapper

(Pristipomoides multidens) and red emperor (Lutjanus sebae).

Morphology Each fish selected for analysis was initially assessed for length (using a mm scale

ruler) and for weight using an electronic spring balance. Each fish was examined for

external abnormalities including lesions or excessive fin damage.

Fish Sampling


FINAL REPORT

Figure 2. Method of demersal fish capture. Baited steel fish trap prior to deployment. In picture: Matthew Badart (left) and Beau Pieterman (right).

Biopsies Collected Fish selected for biopsy (approximately 20 of each target species from each site)

were sacrificed by iki jimi (spike through the brain) and a vacuutainer and needle

were used to collect blood from the caudal vein. These blood samples were allowed

to coagulate and then centrifuged 2400 x g for 10 mins and the serum supernatant

immediately frozen in a cryogenic dry shipper (approx. -190°C). The fish was then

dissected along the ventral line and inspected internally. Many of the fish were

significantly infested with parasites in the body cavity. In red emperor there were

mainly nematodes while in goldband snapper most of the parasites were

encapsulated and adhesive to body organs (liver, gonads, digestive tract). The

following biopsies were collected:

1. Serum samples (see above);

2. Bile was collected from the gall bladder using a 1 ml syringe and frozen at -

20°C;


FINAL REPORT

3. The liver was removed, weighed and subsamples placed in cryogenic vials

and immediately frozen in liquid nitrogen for later analysis;

4. The gonads were removed and weighed. Where available the gonads of 5

male and 5 female fish of each species from each site were preserved in

glutaraldehyde for histology.

Gonad Samples for Histology At each study site (except Site 16), gonads were collected from 5 male and 5 female

individuals of each species (230 samples total). No samples were collected at site 16

due to several additional sites, including site 16, being sampled during the trip, and

all extra preservation vials and solutions were consumed. The samples have been

processed at Curtin University, Ecotoxicology laboratories.

Additional Sampling

Stomach contents

Stomach and intestinal contents were collected from 10 of each species at each

location where available. The stomach contents of the demersal fish were biased due

to the bait placed in the cages. Consequently, the stomach contents was collected for

demersals only if it was identified as ‘other than bait’, The more relevant intestine

contents of the demersal fish, if any, was collected when the stomach was empty. In

total 117 stomach/intestinal content samples were collected.

Flesh Samples

White muscle samples were collected from 10 fish per species per site, and frozen at

- 20ºC

Additional Samples Samples taken in addition to biopsies for analysis at Curtin University were collected

from impacted and reference fishes. Gut contents were collected for 10 goldband

snapper and 10 red emperor at each of the study sites (260 samples total). These

samples are being held at Curtin University until chemical analysis is requested.

Samples of white muscle were also collected from 20 individuals of each species


FINAL REPORT

from each sites. These samples will be stored at Curtin University for one year,

should PTTEP opt for additional chemical or olfactory taint assessment.

Physiological Parameters The condition factor (k) of the fish was calculated as:

where Wg is the gutted weight of the fish and Lf is the fork length. Condition factor

gives an indication of the health status, or ‘fattiness’ of the animal. The liver somatic

index was calculated as

where WL is the liver weight, and gives an indication of the size of the liver relative to

the body size. Similarly the gonado-somatic index (GSI) was calculated as:

where WG is the weight of the gonad. GSI is a measure of the fish’s reproductive

investment.

𝑘𝑘 = �𝑊𝑊𝑔𝑔𝐿𝐿𝑓𝑓3� × 106

𝐿𝐿𝑆𝑆𝑆𝑆 = �𝑊𝑊𝐿𝐿

𝑊𝑊𝑔𝑔� × 100

𝐺𝐺𝑆𝑆𝑆𝑆 = �𝑊𝑊𝐺𝐺

𝑊𝑊𝑔𝑔� × 100

Laboratory Methods


FINAL REPORT

Liver Detoxification Enzymes (EROD activity) In the laboratory, a liver homogenate is centrifuged to isolate the microsomes

containing the detoxification enzymes. These microsomes are then required to

metabolise a model contaminant: ethoxyresorufin. The enzyme metabolising

ethoxyresorufin is therefore named ‘ethoxyresorufin-O-deethylase’ (EROD). The

metabolism of ethoxyresorufin by EROD enzyme activity results in a fluorescent

product, resorufin, which can be measured by fluorimetry at excitation wavelength

530 nm and emission wavelength 585 nm (Hodson et al., 1991). A higher

fluorescence indicates an increased abundance of enzyme that resulted from the fish

assimilating and metabolising high levels of contaminants. The enzyme activity is

reported by unit of protein in the isolated microsome fraction, which normalizes the

data according to the protein density in the liver tissue.

Biliary Metabolites After being processed by liver detoxification enzymes, metabolites are predominantly

eliminated via the bile. However, the bile turnover and by consequence the

concentration of biliary metabolites is influenced by food intake. Therefore, this

biomarker is relevant for recent (weeks) exposure to xenobiotics. The bile of the fish

was collected using a 1 ml syringe, and immediately frozen in liquid nitrogen until

biliary metabolites determination by fixed-wavelength fluorescence (FF)

measurement (Lin et al., 1996). The method is semi-quantitative, and reports

metabolised PAHs as ‘type of metabolites’. The expression ‘type of metabolites’

refers to the various aromatic compounds, most occurring as conjugated metabolites

of PAHs, that are measured in the bile at naphthalene-, pyrene-, or benzo(a)pyrene-

(B(a)P-) specific excitation/emission wavelengths. Fluorescent readings were

performed for naphthalene-type metabolites at excitation/emission 290/335 nm using

1-naphthol (Sigma) as a reference standard. For pyrene-type metabolites and B(a)P-

type metabolites, readings were made using 1-hydroxy pyrene (1-OH pyrene) as a

reference standard at 340/380 nm and 380/430 nm for pyrene- and B(a)P-type

metabolites, respectively. Naphthalene-type metabolites are reported in mg of 1-

naphthol fluorescence units equivalent per mg biliary protein, and pyrene- and B(a)P-

type metabolites are reported in µg of 1-OH pyrene fluorescence units equivalent per

mg biliary protein. Therefore, the biliary metabolite levels observed represent

fluorescence-equivalents of PAH metabolites used as standard.


FINAL REPORT

Sorbitol Dehydrogenase (SDH) activity A blood collection was performed via the caudal artery using a vacuutainer. The

blood was allowed to clot on ice for 15 minutes after which it was centrifuged in a

refrigerated centrifuge at 3000 g for 10 minutes. The serum was immediately

collected and frozen at -80°C until analysis of SDH activity. The quantitation of SDH

activity was determined by the reduction in light absorbance at 340 nm over time.

SDH activity is reported in milli-International Units (mIU), with one U of an enzyme

being defined as the amount of this enzyme which will convert 1 µmol of substrate to

product, per minute.

Gonad Histology Preserved (glutaraldehyde) male and female gonads were washed and transferred to

a 50% ethanol solution for longer term storage. The samples sectioned into 10 mm

cross-sections which were further dehydrated by progressively increasing the ethanol

concentration over four hours. They were then cleared in xylene for a further three

hours before embedding in paraffin wax overnight. These samples were placed in

paraffin wax blocks for sectioning (5 µm) using a microtome. These sections were

fixed to glass slides and gently heated overnight prior to staining. The slides were

stained (Haematoxylin – Eosin) using standard methods (Lillie, 1965). This

differential staining procedure allows the identification of relevant structures in the

tissues (particularly nuclei and membranes) and the identification of any

abnormalities. In gonad tissue it also allows the classification of gametes at different

stages of development.

Histological slides of the ovaries of selected Goldband snapper and Red emperor

females from each site were examined for the development of oocytes. Photographs

of random regions of the slides taken under magnification (x 200) were overlain with

a grid. Fifty grid locations were randomly selected for each slide and the number of

oocytes at each stage (following the scale described by West 1990 and detailed in

Table 2) falling within each grid square was determined. Results are presented as

the average percentage appearance of each stage in the ovary.


FINAL REPORT

Table 2. Scale used for determining oocyte stage in goldband snapper and red emperor.

Oxidative DNA Damage (8-oxo-dG Content) The concentration of the oxidative stress biomarker 8-oxo-7,8-dihydro-2’-

deoxyguanosine (8-oxodG) in the serum samples was measured using a

commercially available enzyme linked immunosorbent assay (ELISA) kit (Trevigen).

Briefly, serum samples were diluted (1:5) with the diluent supplied with the kit and a

supplied 8-oxodG standard was diluted to create a standard curve (0.94 – 60 ng L-1).

These were added to a 96 well microplate which was pre-coated with 8-oxodG with

an 8-oxodG monoclonal antibody. After 1 hour incubation the plate was washed 6

times (Bio-Rad plate washer) and a secondary antibody (horseradish peroxidase

conjugate) was introduced. After further 1 hour incubation the plate was again

washed (6 times) and the plate developed with a trimethylbenzine (TMB) substrate

and the absorbance (450 nm) was measured (Bio-Rad plate reader) after addition of

an acidic stop solution. The intensity of the colour was inversely related to the

amount of 8-oxo-dG in the sample or standard. These were standardised against the

protein content of each serum sample (Lowry et al., 1951).

Statistical Analyses For each variable measured, descriptive statistics including average and standard

errors of the mean have been generated using the program SPSS ver. 20.0. Multiple

comparisons focussed mainly on sites closest to the rig (sites 1, 2, 14, 15) relative to

the reference Site 5, as other sites were mostly sampled in order establish a baseline

for future monitoring of produced formation water discharge. Physiological

parameters condition factor (CF), liver somatic index (LSI) and gonado-somatic index

Stage Name Description Scale

Chromatin nucleolar stage Primordial follicle; large nucleus with single large nucleolus; cell surrounded by few squamous follicle cells. 1

Perinucleolar stage Cytoplasm stains uniformly; multiple nucleuoli appear around nuclear periphery. 2

Cortical alveoli formation Appearance of “yolk” vesicles forming several peripheral rows; oil droplets accumulate in the cytoplasm. 3

Vitellogenic stage Yolk proteins in fluid-filled spheres appear 4

Ripe (mature) stage Nucleus migrates peripherally; yolk granules coalesce and oocyte becomes more transparent; rapid increase in size due to oocyte hydration;

5


FINAL REPORT

(GSI) as well as biomarker levels were compared using a one-way analysis of

variance (ANOVA) after verification of normal data distribution and homoscedasticity.

Post-hoc test Tukey’s-b was applied to identify specific site differences, if relevant. In

cases where data were not normally distributed or heteroscedasticity was observed,

non-parametric multiple comparisons were conducted using the Kruskal-Wallis 1-way

ANOVA followed by a Mann-Whitney U test. For all statistical tests, a significance

level alpha (α) of 0.05 was applied.

All graphs are presented as mean ± standard error of the mean (SEM). Number of

fish for individual means are listed in Table 2 for physiological parameters CF, LSI,

GSI and CF, and according to Table 3 for all biochemical markers except for

oxidative DNA damage where N = 10 fish per site.


FINAL REPORT

Fish Sampled A total of 881 fish were sampled across the 13 study sites (486 goldband snapper

and 395 red emperor) (Table 3). Male and female fish were captured at each study

site with relatively even numbers of both sex collected. Morphological measurements

(lengths, weight, liver weight, sex, gonad weight and stage) were collected for each

of these fish. There were some juvenile fish for which gonadal sex could not be

determined visually.

Results & Interpretation


FINAL REPORT

Table 3. Numbers of demersal fishes sampled during Phase IV of Study S4A.

Goldband Snapper Red Emperor

Total

Site Male Fem Juv Total Male Fem Juv Total

Impacted 1 21 19 0 40 20 20 0 40 80

2 14 15 0 29 17 20 3 40 69

4 11 15 0 26 12 25 2 39 65

8 16 17 0 33 6 11 3 20 53

9 22 17 1 40 10 14 4 28 68

10 29 11 0 40 17 23 0 40 80

11 25 15 0 40 9 14 3 26 66

12 26 15 0 41 8 8 0 16 57

13 26 15 0 41 3 9 1 13 54

14 21 14 2 37 21 18 1 40 77

15 20 19 0 39 15 17 1 33 72

16 17 23 0 40 11 21 2 34 74

Total 210 161 2 373 133 175 13 321 694

Reference 5 20 20 0 40 12 14 0 26 66

Total 58 54 1 113 28 39 7 74 187

Grand Total 268 215 3 486 161 214 20 395 881


FINAL REPORT

Biopsy Collection Biopsies for analysis at Curtin University Ecotoxicology laboratories (serum, bile, liver

and gonad samples) were collected on a total of 519 fish (263 goldband snapper and

256 red emperor) (Table 4). The quota of 20 goldband snapper for biochemical

analysis was reached at all sites but was not reached at 2 sites (12 and 13) for red

emperor. It is not expected that this will markedly affect the outcomes of this study

Table 4. Numbers of fish from which biopsies were collected during the current phase of study S4A.

Site Goldband Snapper Red Emperor Total

Impacted 1 20 21 41

2 20 20 40

4 21 20 41

8 20 21 41

9 20 22 42

10 20 21 41

11 20 20 40

12 20 16 36

13 20 13 33

14 21 21 42

15 21 21 42

16 20 20 40

Total 203 193 396 Reference 5 20 20 40

Total 60 63 123 Grand Total 263 256 519


FINAL REPORT

General Fish Health No gross abnormalities or external parasites were observed on any of the specimen

of goldband snapper or red emperor collected during Phase IV of the study. Internal

parasites were common in both species, with observations of worms adhering to the

external walls of the stomach, intestines or liver (Figure 3). The rate of occurrence of

parasitic worms, and the abundance of worms in the abdominal cavity, seemed to be

similar to those observed in previous samplings.

Physiological Parameters Gross indices issued from physiological measurements can be indicative of effects

following chronic exposure to pollution. While non-specific to individual contaminants,

physiological indices such as condition factor (CF), liver somatic index (LSI) and

gonado-somatic index (GSI) represent a first-level screen to identify if effects have

occurred, and if further investigations are warrant. In this regard, condition factor is a

useful indicator of the ‘fattiness’ of the fish, may provide information on energy

reserves of the fish and possibly the ability of the animal to tolerate toxicant

challenges or other environmental stresses. The liver-somatic index might inform on

liver pathologies, including liver enlargement upon exposure to chronic

contamination. Finally, the gonado-somatic index informs on the reproductive

investment made by fish, as reproduction is often one of the first parameter affected

in fish exposed to contamination (van der Oost et al. 2003).

Figure 3. Parasites commonly found in the abdominal cavity of goldband snapper and red emperor.


FINAL REPORT

Condition Factor The condition factor of goldband snapper collected at three of the 4 sites closest to

the rig was similar to the condition factors in fish originating from the reference site

(Figure 4). Goldband snapper collected at one site (Site 15) located 27 NM north-

west of the rig had higher liver-somatic index relative to reference fish of this species.

The very small difference observed in liver size relative to body size might be a result

of local environmental conditions found at Site 15 e.g. food abundance, as an altered

condition factor is not consistently observed in goldband snapper collected at other

sites close to the rig. Moreover, the condition factor of red emperors collected at all

sites close to the rig did not either differ from the condition factors in red emperors

from the reference site. Condition factor of fish which informs on the general health

status and energy reserves of individuals, might be affected by non-pollutant factors

such as seasons and nutritional levels (van der Oost et al. 2003). It is therefore

concluded that fish of both species collected at the four sites close to the West Atlas

rig are in good physical conditions, as measured by their condition factors.


FINAL REPORT

Figure 4. Condition factors of goldband snapper and red emperors collected at the various study sites during Phase IV of the monitoring study S4A. For both species, fish from all sites had statistically similar CF (p > 0.05 in all cases).

0

1

2

3

1 2 14 15 8 9 4 10 11 16 12 13 5

Cond

ition

fact

or

Sites

Goldband Snapper

0

1

2

3

1 2 14 15 8 9 4 10 11 16 12 13 5

Cond

ition

fact

or

Sites

Red Emperor


FINAL REPORT

Liver Somatic Index The ratio of the liver size relative to body size provides an index that is widely used

as a general indicator of chronic exposure to contaminants. A larger liver can be the

result of exposure to contaminants, in particular contaminants which are largely

metabolised by the liver. However, under field conditions, LSI can also vary

according to biotic or abiotic factors other than pollutants, such as onset of

reproductive activity.

In goldband snapper collected at three of the sites (sites 1, 2 and 15) located close to

the West Atlas well head, the liver size relative to body size was enlarged compared

to the goldband collected at the reference site (Figure 5). In red emperor, a similar

pattern was observed with fish from all sites located close to the well head having

enlarged livers relative to individuals of this species collected from the reference site.

Higher liver size relative to body size has been observed for both species of fish in

previous samplings of these locations. Findings of the present investigation confirm

previous observations (Gagnon and Rawson 2011). While the observation is

consistent through time and species, it is unlikely that larger liver sizes are due to

continuing exposure to contamination as other biomarkers specific to petroleum

hydrocarbons did not confirm exposure of fish to petroleum hydrocarbons at these

locations (see ‘liver detoxification enzymes’ and ‘biliary metabolites’ below). it is

unlikely that enlarged livers are related to reproductive activities, as reproductive

status of fish from all sites were similar (see gonado-somatic index, following

section).

Enlarged livers in fish collected close to the rig might be related to nutritional status of

the fish, in which case increased lipid stores would be deposited in the liver. It is

suggested to investigate liver lipid contents of goldband snapper and red emperors in

future monitoring.

For both species of fish, LSI has shown altered values in previous post-spill

samplings. Liver is a plastic organ with the capability to adjust its mass when

sustained metabolic demand occurs and in this regard, the biological response of

altering hepatic tissue mass might take several months. Short-term biomarkers of

exposure e.g. EROD activity and PAH biliary metabolites are low and suggest no


FINAL REPORT

recent exposure to petroleum hydrocarbons, however it is possible that the liver size

has not fully returned to pre-spill conditions in fish collected close to the well head.

Alternatively, the lower liver size in fish collected close within 27 NM of the rig might

be related to environmental conditions close to the well head, including prey

abundance available to the fish. Abundance of preys might not have returned to pre-

spill event, resulting in lower energy reserved stored in the liver of goldband snapper

and red emperor.


FINAL REPORT

Figure 5. Liver-somatic index in goldband snapper and red emperors captured at study sites in the Timor Sea in November 2011. Bars with different letters indicate statistical differences. Sites 1, 2, 14, 15 were all located within 27 NM of the hydrocarbon release and were considered impacted, while reference Site 5 was located at 110 NM from the West Atlas rig location. LSI at the sites close to the drill rig was different from LSI in reference fish (p < 0.000).

0

5

10

1 2 14 15 8 9 4 10 11 16 12 13 5

LSI

Sites

Goldband Snapper

0

5

10

1 2 14 15 8 9 4 10 11 16 12 13 5

LSI

Sites

Red Emperor

b b b a

b b b b

a


FINAL REPORT

Gonado-somatic Index The GSI, or gonado-somatic index, is a measure of the reproductive competence in

fish. There is increasing evidences that chronic, low-level pollution might impair

reproduction in fish, leading to a long-term decline and potentially, causing local

extinctions of populations (reviewed in Kime, 1995). The gonado-somatic index might

be quite variable between populations, but still represents an indication of the

reproductive status of the fish.

GSI appeared to vary considerably between sites, especially for female goldband

snapper (Figure 6). Differences between sites can be explained by the fact that at

most sites, some female fish had well developed mature gonads ready to spawn

while other female fish were at earlier phases of gonadal development. At other sites,

very few female fish had mature gonads, making the GSI value significantly lower. A

lower GSI at some sites does not imply a negative outcome on reproduction, it rather

informs on the energy directed by the fish up to the capture date to reproductive

maturation of gonadal tissues. To assess if gonad maturation proceeds normally,

histological examination of the gonads has been conducted (see next

section).Similarly for male goldband snapper, individuals at Site 12 were more

advanced in their gonad development relative to fish from the reference site,

however, this does not imply a differential reproductive outcome.

For both male and female adult goldband snapper and red emperors, GSI was

similar in fish close to the well head (sites 1, 2, 14 and 15) relative to GSI in fish

collected at the remote reference Site 5 (Figure 6, Figure 7). The reproductive

investment in gonad development appeared to be comparable in all fish from these

five locations. This confirms the trend observed in previous monitoring of these sites,

where low GSI were initially observed in fish collected close from the well head but

were showing a temporal trend towards similar GSI as in the reference fish.


FINAL REPORT

Figure 6.Gonado-somatic index of male and female goldband snapper captured at study sites in the Timor Sea in Phase IV of the monitoring. Bars with different letters indicate statistical differences. Fish captured at sites within 27 NM of the well head (sites 1, 2, 14, 15) have similar GSI relative to fish from the reference Site 5. GSI in male fish originating from Site 12 is different from GSI in male fish from the reference Site 5 (p =- 0.006). GSI measured in female fish collected at Site 10 is different from GSI measured in female fish from the reference Site 5 (p = 0.001).

0

1

2

3

4

5

1 2 14 15 8 9 4 10 11 16 12 13 5

GSI

Sites

Goldband snapper male

a

0

10

20

30

1 2 14 15 8 9 4 10 11 16 12 13 5

GSI

Sites

Goldband snapper female

b

a

b


FINAL REPORT

Figure 7. Gonado-somatic index of male and female red emperors captured at study sites in the Timor Sea in Phase IV of the monitoring. Male and female fish captured at sites within 27 NM of the well head (sites 1, 2, 14, 15) had similar GSI relative to fish from the reference Site 5 (p > 0.05 in all cases).

0

1

2

3

4

5

1 2 14 15 8 9 4 10 11 16 12 13 5

GSI

Sites

Red emperor male

0

10

20

30

1 2 14 15 8 9 4 10 11 16 12 13 5

GSI

Sites

Red emperor female


FINAL REPORT

Gonad Histology Histological examination of the male gonads revealed no overall differences between

red emperor and goldband snapper at the study sites. Male gonads of each individual

contained spermatagonia at a range of developmental stage with the majority having

spermatagonia containing fully developed sperm. These fish appeared to be either

spawning or about to spawn.

The testes of a single goldband snapper from Site 13 with morphologically male

gonads contained ovarian tissue (Figure 8). The testicular tissue was interspersed

with oocytes at varying stages of development. While such an occurrence can be due

to a contamination event (e.g., Jobling et al 1998), there is no evidence to suggest

such a link in this case. The occurrence of simultaneous hermaphroditism (the

presence of a male and a female gonad in the same individual) is not unknown in

fishes (e.g., some Serranidae). Other species (e.g., some Sparidae) occur as

rudimentary hermaphrodites (either having both male and female gonads or gonads

with both male and female tissue in early life stages then developing in favour of a

single gender with maturity). The occurrence of intersex gonads (the presence of

oocytes in a testis) has also been reported as naturally occurring (e.g., Korner et al.

2005). As far as we are aware this is the first time this has been observed in

goldband snapper. Throughout study S4A, gonads from over 150 male goldband

snapper have been examined histologically and this is the first occurrence of the

phenomenon. Given the rarity of this occurrence and the remoteness of the location

from any known source of contamination (relative to other sites nearer the Montara

well head), it appears that for this species the presence of ovarian tissue in the testes

is normal, though uncommon.

The ovaries of female goldband snapper and red emperor examined histologically

were without apparent pathology. In general the goldband snapper gonads examined

were less well developed than the red emperor with a greater number of individuals

lacking stage V oocytes. At sites 15, 11 and 5 none of the goldband snapper

examined contained stage V oocytes and at each of the other sites there was at least

1 individual examined with a similar condition (Figure 9).


FINAL REPORT

Figure 8. Histological (HE stain) examination of intersex gonad of morphologically male goldband snapper collected from Site 13. Inset shows characteristic lobed structure of male testis (note the mature sperm in the efferent duct). ED = Efferent Duct, S = Sperm (with developmental stage indicated), O = oocyte (with developmental stage indicated).

At all sites, there were red emperor, the ovaries from which contained stage V

oocytes. This suggests that while there is significant variation within the populations

of each species, the red emperor were closer to their optimal spawning time. There

was no indicative trend in the presence of stage V gonads associated with the

location of the Montara well head. While Site 15 is in close proximity, Sites 11 and 5

are located at a greater distance.

There some between-site variation within each species as to both the dominant

oocyte stage present and the furthest oocyte development observed (both are

commonly used metrics for determining differences between individuals). The

goldband snapper captured in the sites closest to the Montara well head had oocyte

stage distributions which were not dissimilar to those at sites further away. Of all the

goldband snapper collected, only those from Site 8 had an increased proportion of

O IV-V

O III

O II

ED

S IV

S III


FINAL REPORT

Stage I oocytes, indicating that these fish may be later in their reproductive cycle

than those at other sites.

Red emperor collected at sites 8 and 9 had an increased proportion of stage I and II

oocytes with a decreased proportion of stage III oocytes compared to individuals

from other study sites. Sites 8 and 9 were the furthest north east of the Montara well

head. This result does not appear related to the Montara release. It is likely a

geographically driven difference in spawning time.


FINAL REPORT

Figure 9. Percent composition of ovaries of goldband snapper and red emperor from Timor Sea study sites by Oocyte stage. Oocyte stages defined and described given in Table 2.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1 2 14 15 8 9 4 10 11 12 13 5

% O

ocyt

es S

tage

Site

Goldband snapper

Stage 5Stage 4Stage 3Stage 2Stage 1

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1 2 14 15 8 9 4 10 11 12 13 5

% O

ocyt

es S

tage

Site

Red emperor

Stage 5Stage 4Stage 3Stage 2Stage 1


FINAL REPORT

Biochemical Parameters

Liver Detoxification Enzymes Liver detoxification enzymes are part of a detoxification system found in most

animals. Upon exposure to certain classes of contaminants such as petroleum

hydrocarbons, polychlorinated biphenyls, etc., liver enzymes are induced at

high levels in order to proceed with oxidation of xenobiotics which aims to

eliminate the chemical out of the body. Specific detoxification enzymes, such

as ethoxyresorufin-O-deethylase (EROD), are measurable in fish livers even if

the inducing chemical is well below detectable levels in the environment

(Landis and Yu, 1995).

Liver detoxification enzymes are present with relatively low activity in normal

animals as these enzymes also fulfil several metabolic functions. In fact,

activity of the liver enzymes can vary naturally with sexual hormones,

increasing the variability of this biomarker. For example, the presence of

estrogen in the reproductively active female fish can significantly inhibit the

measured EROD activity levels (van der Oost et al., 2003). Because of this

potential alteration by reproductive hormones, liver detoxification activity has to

be considered separately for male and female organisms.

Because liver enzymes do perform normal metabolic activities, it is expected to

measure background enzymatic activity levels which are specific to each

species. Concerns arise when activity levels of enzymes closely correlated to

exposure to contaminants e.g. EROD activity, is increased by 2-fold or more in

a consistent manner across contaminant-exposed animals.

Previous investigations carried out following the Montara incident revealed that

goldband snapper and red emperor collected in close proximity to the West Atlas rig

exhibited elevated hepatic EROD activity, suggesting that these fish were exposed

to, had assimilated, and were in the process of metabolising petroleum hydrocarbons

(Gagnon and Rawson 2011). In Phase IV investigation, liver detoxification activity in

both species of fish, and for both sexes, were similar in fish collected close to the well

head relative to the EROD activity in fish from the reference area (Figure 10, Figure


FINAL REPORT

11). This result suggests that fish collected in close proximity of the well head are no

longer exposed to the inducing compound which triggered EROD activity levels in

past studies. It also provides background activity levels and variability for

comparative purposed in future studies.

It is noted that male red emperor collected at Site 10 had a significantly higher liver

detoxification activity relative to male red emperor from the reference Site 5 (p <

0.001). High EROD induction suggests that these fish are exposed to compounds

inducing EROD activity, which might originate from natural seepage.

This result supports observations from previous samplings where several individuals

collected at Heywood Shoal exhibited high EROD activity and biliary metabolite

levels. A similar situation might occur at Site 8.

Similar conclusions were reached when a three-year fish health monitoring study was

conducted following the 2002 Prestige oil spill that occurred off the coast of Spain.

Biomarkers of fish health measured in the Spanish study included EROD activity,

with measured levels returning to baseline levels after two to three years post-spill

(Martinez-Gomez et al. 2009). The Prestige oil involved a heavy fuel oil (M-100) of

different composition to the Montara oil, however both crude oils had a low content of

high molecular weight polycyclic aromatic hydrocarbons (PAHs) (Alzaga et al. 2004).

In contract, the cold environment in which the heavy fuel oil from the Braer spill

(Shetland Islands, United Kingdom, 1993) was discharged was still a major concern

6 years after the event (McGenity et al. 2012). Generally, crude oils with a low

content of high molecular weight PAH are prone to rapid biodegradation in marine

tropical waters (McGenity et al. 2012).


FINAL REPORT

Figure 10. EROD activity (pmol/mg protein/min) in male and female goldband snapper captured as study sites in the Timor Sea in November 2011. EROD activity in fish collected within 27 NM of the rig (sites 1, 2, 14, 15) is not statistically different (p = 0.419 and 0.700 for male and female, respectively) form EROD activity in fish from the reference Site 5. Other investigated sites were not statistically different from the reference Site 5.

0

10

20

30

40

1 2 14 15 8 9 4 10 11 16 12 13 5

ERO

D a

ctiv

ity

Sites

Goldband snapper male

0

10

20

30

40

1 2 14 15 8 9 4 10 11 16 12 13 5

ERO

D a

ctiv

ity

Sites

Goldband snapper female


FINAL REPORT

Figure 11. EROD activity (pmol/mg protein/min) in male and female red emperor captured as study sites in the Timor Sea in November 2011. Bars with different letters indicate statistical differences. EROD activity in fish collected within 27 NM of the rig (sites 1, 2, 14, 15) is not statistically different (p = 0.169 and 0.605 for male and female, respectively) form EROD activity in fish from the reference Site 5. Site 8 is also significantly different form the reference Site 5 (p < 0.001 and p = 0.003 for male and female respectively).

0

25

50

75

100

1 2 14 15 8 9 4 10 11 16 12 13 5

ERO

D a

ctiv

ity

Sites

Red emperor male b

a

0

25

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100

1 2 14 15 8 9 4 10 11 16 12 13 5

ERO

D a

ctiv

ity

Sites

red emperor female

a

b


FINAL REPORT

Biliary Metabolites Following oxidation of the chemical by the liver detoxification enzymes, the

metabolites are directed to the biliary secretions for elimination out of the body via

the intestinal route. The liquid bile secretions can therefore accumulate metabolites

at levels up to 1000x more concentrated than in the surrounding environment (Hellou

and Payne 1987; Meador et al. 1995), making this biomarker a very sensitive

indicator of exposure to petroleum hydrocarbons.

As most vertebrates, fish do not accumulate petroleum hydrocarbons in their flesh

because they are capable of metabolising PAHs at rates that prevent significant

bioaccumulation (Hartung, 1995). Enzymes responsible for the metabolisation of

PAHs do not appear to be affected by the gender of the animal and consequently,

biliary metabolite levels can be combined for both male and female fish of one

species. The elimination of PAH metabolites is evaluated by the measurement of

three types of metabolites in the biliary secretions, these being naphthalene, pyrene

and benzo(a)pyrene [B(a)P] metabolites.

In previous sampling following the Montara incident, both species of fish collected in

proximity of the West Atlas well head have shown elevated PAH biliary metabolite

levels relative to reference fish, in at least one occasion (Gagnon and Rawson,

2011). In Phase IV of the study however, goldband snapper and red emperor

collected at sites within 27 NM from the well head no longer exhibited elevated PAH

biliary metabolite levels relative to reference fish (Figure 12, Figure 13). This result,

along with the low liver detoxification activity, further supports the postulate that fish

are no longer exposed to petroleum hydrocarbons from the damaged underwater

infrastructures.

At site 16, goldband snapper consistently showed elevated pyrene and BaP biliary

metabolites. This site is located along one of the Heywood Shoal ridges, and is in

close proximity to the known natural Cornea seep (Brunskill et al., 2011). The

presence of this seep might be related to the ongoing positive biomarker responses

observed in fish collected at Heywood Shoal.


FINAL REPORT

Figure 12. Levels of naphthalene- (mg/mg protein), pyrene- (µg/mg protein) and BaP (µg/mg protein) -type biliary metabolites in goldband snapper captured at study sites in the Timor Sea in November 2011. Bars with different letters indicate statistical differences. Goldband snapper collected from study sites located within 27 NM from the well head had similar biliary metabolite levels (p =0.056, 0.062 and 0.454 for naphthalene, pyrene and BaP-type metabolites respectively) relative to fish originating from reference Site 5.

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FINAL REPORT

Figure 13. Levels of naphthalene- (mg/mg protein), pyrene- (µg/mg protein) and BaP (µg/mg protein) -type biliary metabolites in red emperor captured at study sites in the Timor Sea in November 2011. Red emperor collected from study sites within 27 NM from the well head had similar biliary metabolite levels (p =0.166, 0.522 and 0.872 for naphthalene, pyrene and BaP-type metabolites respectively) relative to the fish collected at the reference Site 5.

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FINAL REPORT

Sorbitol Dehydrogenase (SDH) Activity The enzyme sorbitol dehydrogenase is primarily found in the liver and catalyses the

reversible oxidation-reduction reaction between fructose and sorbitol. Its involvement

in energy metabolism is biologically relevant for metabolic purposes. In addition, the

presence of SDH in the bloodstream is informative of liver damage which might

follow exposure to contamination (Heath 1995). Previous work has shown that such

increases in SDH in the bloodstream can follow exposure to organic contaminants

including petrogenic compounds (e.g., PAHs) (Ozetric and Krajnovic-Ozetric, 1993).

SDH activity is not affected by conditions such as sex of the animals or reproductive

status which often are confounding factors in the interpretation of other biomarkers.

In previous investigations on the effects of the Montara incident on fish health, levels

of SDH activity in the bloodstream of goldband snapper was at similar levels in

exposed snapper as it was in reference fish of this species. However, immediately

following the Montara hydrocarbon release, red emperor collected in impacted areas

during in Phase I (November 2009) showed a significant increase in SDH activity in

the blood serum, suggestive of hepatocellular damage related to exposure to

petroleum hydrocarbons. The elevated SDH levels in red emperor were not observed

in subsequent sampling events, indicating that liver functions in fish collected in

impacted areas have returned to reference levels within 4 months post-control of the

well release.

In Phase IV of the study (November 2011), sorbitol dehydrogenase activity in

goldband snapper and red emperor remained comparable in fish collected from sites

close form the West Atlas location and in fish collected from the reference area

Figure 14). This result is in agreement with previous samplings performed at these

sites, and indicates that the Montara incident has not resulted in chronic hepatic

damage in fish collected within 27 NM from where the hydrocarbon release occurred.


FINAL REPORT

Figure 14. SDH activity (mIU) in goldband snapper and red emperor collected from study sites in the Timor Sea in November 2011. Both species of fish collected in the vicinity of the rig (sites 1, 2, 14 15) had comparable SDH activity levels to the fish collected at the reference Site 5 (p = 0.330 and 0.101 for goldband snapper and red emperor respectively).

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FINAL REPORT

DNA damage As noted in previous phases of Study S4A the levels of oxidative DNA damage was

between 2 and 4 times higher in goldband snapper than red emperor across all sites.

This is an indication of a physiological difference in either the rate of damage and

endogenous repair that occurs in these fish. It is not a difference in their susceptibility

to increased oxidative damage.

There were no differences between the levels of oxidative damage in goldband

snapper between the study sites (Figure 15). Statistically, there were overall

differences between the oxidative DNA damage in red emperor at the study sites.

However, there were no trends in these differences which were associated with the

location of the Montara well head. The mean oxidative DNA damage in red emperor

at the reference location was close to the overall mean with no detectable differences

between this and any other study site. Importantly, none of the sites closest to the

Montara well head, had oxidative DNA damage levels which were above those

measured in fish from the reference location (Site 5).


FINAL REPORT

Figure 15. Oxidative DNA damage (ng/ml/mg protein) in goldband snapper and red emperor at study sites in the Timor Sea in November 2011. No statistical differences were identified between goldband snapper collected at the study sites (p = 0.662). Between site differences were detected between red emperor collected between sites (p = 0.036) but no differences were detected between the reference site (Site 5) and any of the other study sites.

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FINAL REPORT

The Montara drill rig located in the Timor Sea suffered a well head accident in August

2009, resulting in the uncontrolled discharge of oil and gas condensate. The

discharge commenced on 21st August 2009 and was stopped on 3 November 2009.

An estimated 23,000 barrels of oil and gas condensate was released to the marine

environment, coinciding with the onset of the reproductive season for the

commercially important goldband snapper and red emperor.

To assess the long term potential effects of hydrocarbon exposure to fish,

investigations commenced immediately after the control of the hydrocarbon release

and continued for two years thereafter. To date, four sampling events have been

conducted, collecting a total of 1662 fish.

The first three phases of the study found that immediately following the incident, fish

collected in close proximity to the well head exhibited signs of exposure and effects

however a temporal decline of effects was observed during the 12 months post-spill.

While increased levels of oxidative DNA damage and increased liver size were still

observed at some sites, the magnitude of the effects relative to reference areas was

reduced, suggesting an ongoing trend towards normal, reference conditions.

This report describes the results of Phase IV of the monitoring program (November

2011). Two years following the end of the spill, biomarker levels in goldband snapper

and red emperor had mostly returned to reference levels with the exception of the

Conclusions


FINAL REPORT

liver somatic index (i.e. liver size relative to body size) which remained elevated in

both species collected within 27 NM of the well head, relative to reference fish.

Other biomarkers of exposure such as liver detoxification enzymes and PAH biliary

metabolites do not indicate recent exposure of these fish to petroleum hydrocarbons,

which could have been related to this increased liver somatic index. It is possible that

this long-term biomarker of exposure to contaminants has not yet returned to

background conditions, or alternatively an increased liver size relative to body size

could be related to environmental conditions prevailing at these sites.

Phase IV of the monitoring program included a number of first-time sites located 30

to 80 NM form the well head. The aim of fish collection at these sites was two-fold.

Firstly, sites 12, 13, 14 and 15 were sampled to provide a baseline for evaluating

exposure and possible impacts on fish health, if any, once petroleum production and

discharge of produced formation waters has commenced at the offshore platform. In

addition to baseline biomarker levels, the sampling of these sites provided important

information on the natural variability of physiological measurements and biomarker

measurements that can be expected in the absence of contamination.

Secondly, newly selected sites 10, 11 and 16 were all in all in close proximity to the

previously sampled Heywood Shoal (Site 4), and were added to further investigate

positive biomarker responses measured in fish collected at this site during previous

phases of the monitoring program. The possibility that fish collected at Heywood

Shoal form a single fish assemblage is recognised, as the four sampling locations are

geographically close and bathymetrically connected. Nevertheless, the four sites

were discretely sampled. It was found that male red emperor collected from Site 10

showed elevated liver detoxification relative to fish collected close to the Montara well

head, as well as relative to reference fish. At site 16, goldband snapper had elevated

PAH biliary metabolites, indicating that fish were exposed to, and had assimilated

and metabolised petroleum hydrocarbons. It is known that the Cornea seep exists in

close proximity to site 16 (Brunskill et al., 2011).

Overall, measurements of physiological parameters and biomarkers of fish health in

goldband snapper and red emperor two years following the Montara incident indicate

that only one physiological measurement, liver-somatic index, remain elevated in fish

collected within 27 NM of the drill rig. However, all other physiological parameters as

well as biomarkers of fish health measured in these two species had returned to


FINAL REPORT

reference levels. Phase IV of the monitoring program provided baseline data and

informed on natural variability of biological parameters for future monitoring once

production and PFW discharge have started at the new offshore facility.


FINAL REPORT

The fish health monitoring program conducted since 2009 has established that

immediately following the Montara well release, the commercially important goldband

snapper and red emperor collected within 80 NM of the well head were exposed to,

and assimilated petroleum hydrocarbons. Fish collected within 20 NM from the well

head in past surveys, had an increased liver size and occasionally, increased

oxidative DNA damage. The present survey conducted two years following the

incident shows that most physiological and biochemical markers measured in fish

collected close to the well head have returned to reference levels. However, liver size

relative to body size was significantly larger in fish collected close to the well head.

The deployment of the new processing facility to the Montara field will generate

produced formation water (PFW) discharges which contain dissolved petroleum

hydrocarbons as well as other industrial compounds. PAHs originating from PFW

discharge can be found in fish collected in the proximity of petroleum production

facilities (Gagnon, 2011). Therefore, the following recommendations are made:

Recommendation 1: The monitoring of fish health in the vicinity of

the processing facility utilising biomarkers of exposure and effects in

goldband snapper and red emperor should continue. This will

provide critical information on the impact/no impact of PFW

discharge on the local environment.

Recommendation 2: Additional samples of water and sediments

should be collected in the vicinity of the processing facility to assess

if PFW discharge contributes significantly to the environmental load

of PAHs.

Recommendations


FINAL REPORT

Recommendation 3: Yearly monitoring should be conducted until

all of the biomarkers of fish health have stabilised. Subsequent

monitoring should be performed biennially.

Recommendation 4: If significant process changes affecting the

qualitative or quantitative characteristics of discharged PFW occurs,

annual monitoring should be re-established.


FINAL REPORT

Alzaga R, Montuori P, Ortiz L, Bayona JM, Albaigés J, 2012. Fast solid-phase

extraction–gas chromatography–mass spectrometry procedure for oil fingerprinting:

Application to the Prestige oil spill, J. Chomatogr. 1025:133-138

Brunskill GJ, Burns KA, Zagorskis, 2012. Natural flux of greenhouse methane from

the Timor Sea to the atmosphere. J. Geophys. Res. 116 doi:10.1029/2010JG001444.

Gagnon MM (2011) Evidence of exposure of fish to produced water at three offshore

facilities, North West Shelf, Australia. In: Lee K, Neff J (Eds) Produced water:

environmental risks and advances in mitigation technologies. Springer, New York,

pp. 295–310.

Gagnon MM, Hodson PV, 2012. Field studies using fish biomarkers - How many

fish are enough? Mar Poll Bull, DOI: 10.1016/j.marpolbul.2012.08.016

Gagnon MM, Rawson C, 2011. Montara Well Release, Monitoring Study S4A –

Assessment of Effects of Timor Sea Fish. Curtin University, Perth, Australia, 208

pages. http://www.environment.gov.au/coasts/oilspill/scientific-monitoring.html#four

Hartung R., 1995. Assessment of the potential for long-term toxicological effects of

the Exxon Valdez oil spill on birds and animals. IN: Exxon Valdez oil spill: Fate and

Effects I Alaskan waters, ASTM, pp. 693-725.

Heath AG, 1995. Water Pollution and Fish Physiology 2nd ed. Florida: CRC Press.

359 p. Hellou, J., Payne, JF, 1987. Assessment of contamination of fish by water-soluble

fractions of petroleum: a role for bile metabolites. Environ Toxicol Chem. 6: 857-

862.

References


FINAL REPORT

Jobling S, Nolan M, Tyler CR, Brighty G, Sumpter JP, 1998. Widespread sexual

disruption in wild fish. Environ Sci Tech, 32:2498-2506

Kime, DE, 1995. The effects of pollution on reproduction in fish. Rev. Fish Biol.

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Korner O, Vermeirssen ELM, Burkhardt-Holm P, 2005. Intersex in feral brown trout

from Swiss midland rivers, J Fish Biol, 67(6): 1734-1740

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chemicals upon ecological systems. Lewis, Boca Raton, Florida.

Lillie RD, 1965. Histopathologic Techniques and Practical Histochemistry, 3rd

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Lin ELC, Cormier SM, Torsella JA, 1996. Fish biliary polycyclic aromatic

hydrocarbon metabolites estimated by fixed-wavelength fluorescence: comparison

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the Folin reagent. J Biol Chem. 193: 265-275.

Martinez-Gomez C, Fernandez B, Valdes J, Campillo JA, Benedicto J, Sanchez F,

Vethaak AD, 2009. Evaluation of three-year monitoring with biomarkers in fish

following the Prestige oil spill (N Spain). Chemosphere. 74: 613-620.

McGenity TJ, Folwell BD, McKew BA, Sanni GO, 2012. Marine crude oil

biodegradation: a central role for interspecies interactions. Aquat Biosyst

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Meador JP, Stein JE, Reichert WL, Varanasi U, 1995. Bioaccumulation of polycyclic

aromatic hydrocarbons by marine organisms. Rev Environ Contam Toxicol. 143:79-

165.

Ozretic B, Kranovic-Ozretic M. 1993. Plasma sorbitol dehydrogenase, glutamate

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FINAL REPORT

Appendix I – Comments and Authors’ Responses

Comment Response See Pg

1 Why were gonad samples not

collected at Site 16?

Explanatory text added: “No

samples were collected at site 16

due to several additional sites,

including site 16, being sampled

during the trip, and all extra

preservation vials and solutions

were consumed.”

24

2 Re: suggestions of future

monitoring of liver lipid content in

subsequent samplings; Refer to

future monitoring in later section

of the report

A “Recommendations” section

has been added to the report.

Recommendations 3 and 4 relate

to future monitoring.

60

3 Re: suggestions of future

monitoring of the EROD

biomarker in goldband snapper

and red emperor.

As for Comment 2 above. 60

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